Asymmetric Introgression and Cryptic Natural Hybridization between Two Species of Teucrium Section Polium (Lamiaceae) on the Balkan Peninsula.

In this work, we analyzed the morphology and genetic structure of Teucrium montanum, T. capitatum and their hybrid T. × rohlenae from three syntopic populations. A morphometric study showed that the parents and their hybrids exhibited continuous morphological variation, with the hybrid positioned exactly between the parents. Genetic analysis revealed that plants morphologically identified as T. × rohlenae are fertile hybrids that produce hybrid swarms dominated by later-generation hybrids. This suggests that introgression, rather than speciation, is the more likely outcome of hybridization between these plant species. The extent and direction of gene flow between the two species differed markedly between the three syntopic localities. At the Trilj locality, it was clearly unidirectional, with T. capitatum playing the dominant role. At the Sicevo locality, gene flow was slightly asymmetric, favoring the genetic background of T. capitatum, while at the Sliven site, it was completely asymmetric in the opposite direction. The extreme case of unidirectional gene flow was observed at the Trilj locality where plants morphologically identified as T. montanum could not be genetically distinguished from T. capitatum. This suggests that interspecific hybridization occurred long ago, leading to introgression and cryptic hybrids, blurring of species boundaries and generating evolutionary noise.

Phytogeographic Characteristics of Montane Coniferous Forests of the Central Balkan Peninsula (SE Europe).

We investigated taxonomic and endemic richness, patterns of spatial distribution, cenotic and spatial diversification, and chorological and life form spectra of montane coniferous forests in the central part of the Balkan Peninsula. We collected information on 1435 taxa (1351 at the level of species and 84 subspecies) with 65,289 species-occurrence data, published in 1930 original plots with a total area of about 215 ha in the analysis. All statistical analyses (univariate and multivariate) were performed on binary matrices prepared for different levels of analysis. Our main results showed that the montane coniferous forests of the central Balkan Peninsula represent very species-rich vegetation. At the same time, the high proportion of endemics indicated that the montane coniferous forests of the central Balkan Peninsula differ significantly from Central European and boreal forests of a similar type. Furthermore, we found that there were regional differences in the species composition of the coniferous forests of the Balkan Peninsula, and that the primary centers of floristic richness are located in the area of the central and continental Dinarides. This latter finding suggested that the true centers of the richness of European coniferous forests are located south of the Limestone Alps-Western Dinarides-Carpathian Foothills line in Romania, which used to be considered the center of the richness of the coniferous forests in Europe.

Influence of Habitat Types on Diversity and Species Composition of Urban Flora-A Case Study in Serbia.

The aim of this study was to investigate the floristic composition and diversity of seven urban habitat types in 24 Serbian cities with different climatic affiliation. In each of the 24 cities, we selected 1 ha plots representing a habitat from one of the following groups: square, boulevard, residential area with compact and with open building pattern, city park, and sites with early and mid-succession vegetation stages. All vascular plant species that occur spontaneously in these plots were observed. Data on the main climatic characteristics were collected for each plot, and data on the life forms were obtained for each species recorded. Diagnostic species were identified for each habitat type analyzed, and alpha, beta and gamma diversity were calculated. A total of 674 taxa were recorded in the studied area. Significant differences were observed in habitats by diagnostic species and by life form representation. The lowest alpha and gamma diversity and the dominance of therophytes were observed in habitat types with intensive anthropogenic impact, whereas the highest number was recorded in mid-successional sites and residential areas with a compact building pattern. The analysis showed that habitat type influences species composition much more than climate.

Understanding biogeographical patterns in the western Balkan Peninsula using environmental niche modelling and geostatistics in polymorphic Edraianthus tenuifolius.

The Balkan Peninsula represents one of the three southern European glacial refugia where biodiversity persisted throughout the climatically unstable Quaternary. This study considered the 'refugia within refugia' model, which assumes the environmental heterogeneity over time and space in larger refugia. To better understand patterns shaped during the Quaternary climatic oscillations, the present and last glacial maximum (LGM) environmental conditions and current morphological variability of Edraianthus tenuifolius, an endemic plant of the western Balkans with a well-known therphical structure, were considered. Potential present and LGM distributions were studied through environmental niche modelling using 161 data of occurrences and six bioclimatic variables, hindcasting the model to LGM conditions using three different global circulation models. To test the geographical variability of the reproductive region, 41 characters of 667 inflorescences from 35 populations within the distribution range were measured. Geographical patterns, using geostatistics together with univariate and multivariate statistical approaches, were analysed. The environmental niche model suggested the current potential distribution in correspondence to its known occurrences. The hindcast to LGM conditions suggested two separate areas of environmental suitability, one in the present-day northern Adriatic coast of Croatia (Istrian Peninsula, Kvarner) and another on the present-day south-eastern Adriatic coast (southern Dalmatia, Montenegro and northern Albania). Morphological variability showed a similar pattern, where southern populations separated from northern populations according to a major split in the central part of its distribution range (central Dalmatia). On other levels, stronger barriers were predicted to separate northern populations from the eastern Istrian Peninsula and the Kvarner area. The results suggested congruent biogeographical patterns to the already known phylogeographical structure. Both environmental niche modelling and the geographical variability of morphological characters suggested spatial partitioning, indicating the potential presence of two separate refugia during the LGM.

Habitat-related variation in composition of the essential oil of Seseli rigidum Waldst. & Kit. (Apiaceae).

Plant specialised metabolites like essential oils are highly variable depending on genetic and various ecological factors. The aim of the present work was to characterise essential oils of the species Seseli rigidum Waldst. & Kit. (Apiaceae) in various organs on the individual and populational levels. Geographical variability and the impact of climate and soil type on essential oil composition were also investigated. Individually sampled essential oils of roots, aerial parts and fruits of plants from seven populations were analysed by GC-FID and GC-MS. The investigated populations showed high interpopulational and especially intrapopulational variability of essential oil composition. In regard to the variability of essential oils, different chemotypes were defined. The essential oils of S. rigidum roots represented a falcarinol chemotype, oils of aerial parts constituted an α-pinene or α-pinene/sabinene chemotype and fruit essential oils can be characterised as belonging to a complex sabinene/α-pinene/ß-phellandrene/falcarinol/germacrene B chemotype. At the species level, analysis of variance (ANOVA), principal component analysis (PCA) and canonical discriminant analysis (CDA) showed that the plant part exerted the strongest influence on the composition of essential oils. Climate had a high impact on composition of the essential oils of roots, aerial parts and fruits, while influence of the substrate was less pronounced. The variations in main compounds of essential oils based on climate or substrate were complex and specific to the plant part.

Chemical Composition and Antimicrobial Activity of the Essential Oil and Methanol Extract of Hypericum aegypticum subsp. webbii (Spach) N. Robson.

Hypericum aegypticum subsp. webbii is an evergreen shrub spread in Mediterranean part of central and southeastern Europe. The chemical composition and antimicrobial activity of the essential oil and MeOH extract of H. aegypticum subsp. webbii were investigated. The monoterpenes α-pinene (63.4 - 68.5%) and ß-pinene (16.9 - 17.0%) were main compounds in the volatile oil from aerial parts. In the cluster analysis, the essential oil of H. aegypticum subsp. webbii was separated and chemically different from the oil of other subspecies of H. aegypticum as well as other Hypericum species from Greece. SIMPER analysis revealed that α-pinene (24.79%) was the component that contributed the most to differences between all oils. Also, there was extremely high overall dissimilarity between three subspecies of H. aegypticum. MeOH extract of aerial parts of H. aegypticum subsp. webbii contained flavonoids rutin (56.4 ± 0.9 mg/g), hyperoside and quercetin, and phenolic acids chlorogenic and caffeic acid, while naphthodianthrones were not detected. The antimicrobial activity of essential oil was moderate (MIC from 100 to >200 µg/ml), while MeOH extract inhibited the growth of Gram-positive bacteria Bacillus subtilis, Enterococcus faecalis, Staphylococcus epidermidis, and Micrococcus luteus (MIC 50 - 100 µg/ml), more pronounced than the extract of H. perforatum (MIC 200 - >200 µg/ml).

Seasonal variations in the composition of the essential oils of Lavandula angustifolia (Lamiacae).

Seasonal variations in the composition of the essential oils obtained from the same individual (of the same genotype) of Lavandula angustifolia cultivated in Belgrade were determined by GC and GC/MS. The main constituents were 1,8-cineole (7.1-48.4%), linalool (0.1-38.7%), bomeol (10.9-27.7%), beta-phellandrene (0.5-21.2%) and camphor (1.5-15.8%). Cluster analysis showed that the 21 samples collected each month during the vegetation cycle were separable into three main clades with different compositions of essential oils. In the shoots with flowers, inflorescences and fruits of clade I, linalool is dominant, in the young leaves before flowering and old leaves of clade II, 1,8-cineole is dominant. In the young and incompletely developed leaves of clade III, beta-phellandrene is dominant. The composition of the essential oils of lavender depended on the plant part and the stage of development.

Variability of the root essential oils of Seseli rigidum Waldst. & Kit. (Apiaceae) from different populations in Serbia.

The chemical compositions of the essential oils of seven natural populations of Seseli rigidum were analyzed. The essential-oil yield ranged from 0.16 to 2.09%. Analysis of variance (ANOVA) revealed that there were no statistically significant differences in the mean essential-oil yields between the populations, and no significant influence of the climate or soil type on the oil yield was observed. In all 67 analyzed samples, the polyacetylene falcarinol was the main compound, followed by octanal, methyl linoleate, α-muurolene, 3-butylphthalide, falcarinone, muurola-4,10(14)-dien-1ß-ol, ß-sesquiphellandrene, salvial-4(14)-en-1-one, δ-amorphene, spathulenol, and isospathulenol. The principal component analysis (PCA), the canonical discriminant analysis (CDA), and the cluster analysis (CA) revealed differentiation between the populations based on the climate. Three groups of populations were formed; the first group was composed of samples growing in regions with a humid climate, with oils having high falcarinol and low sesquiterpene contents, and the second and third groups comprised samples exposed to semi-arid climate, with oils characterized by a lower falcarinol and higher α-muurolene, δ-amorphene, ß-sesquiphellandrene, and salvial-4(14)-en-1-one contents. The semi-arid populations were divided into two groups, which were distinguished based on the oil contents of sesquiterpenes, falcarinone, and 3-butylphthalide. On the other hand, no clear separation between populations based on the different soil types could be observed.

Seasonal variations in the composition of the essential oils of rosemary (Rosmarinus officinalis, Lamiaceae).

Seasonal variations in the composition of the essential oils obtained from rosemary plants of the same genotype cultivated in Belgrade were determined by GC and GC/MS. The main constituents were camphor (18.2 - 28.1%), 1,8-cineole (6.4-18.0%), alpha-pinene (9.7-13.5%), borneol (4.4-9.5%), camphene (5.1-8.7%), beta-pinene (2.1-8.1%), beta-phellandrene (4.6-6.5%), myrcene (3.4-5.9%) and bornyl acetate (0.2-7.9%). Cluster analysis showed that 16 samples that had been collected each month during the vegetative cycle can be separated into three main clades with different compositions of essential oils. In the shoots with fruits ('fruits' - Clade I) and shoots with developed leaves ('old shoots' - Clade III) camphor is dominant. In shoots with young and incompletely developed leaves ('young shoot' - Clade II) camphor and 1,8-cineole had almost the same concentration. The fact that the same genotype during the growing seasons can synthesize oils that are so different that they can be classified as different chemotypes confirms the opinion that the chemical composition of essential oils sometimes critically depends on the time of collection. Also, for the definition of chemotypes it is not enough to base this on a chemical analysis of an oil from one phenophase only.

Environment-related variations of the composition of the essential oils of rosemary (Rosmarinus officinalis L.) in the Balkan Penninsula.

Composition of the essential oils of Rosmarinus officinalis of ten populations from the Balkan Peninsula were determined by GC/FID and GC/MS. The main constituents were 1,8-cineole, camphor, α-pinene, and borneol. Multivariate statistical analysis (UPGMA cluster analysis and principal-component analysis (PCA)) revealed two major types of rosemary oil, i.e., 1,8-cineole and camphor-type, and two intermediate types, i.e., camphor/1,8-cineole/borneol type and 1,8-cineole/camphor type. The regression analyses (simple linear regression and stepwise multiple regression) have shown that, with respect to basic geographic, orographic, and 19 bioclimatic characteristics of each population, bioclimatic factor temperature of habitat represented the dominant abiogenetic factor, which, in chemical sense, led to differentiation of populations in the studied region. Also, the regression analysis have shown that some constituents of essential oils are independent of any single bioclimatic factors. However, some constituents display statistically significant correlations with some abiotic factors.

Environmental and seasonal impacts on the chemical composition of Satureja horvatii Silic (Lamiaceae) essential oils.

The chemical composition of the essential oils of Satureja horvatii Silic from two natural habitats (Mt. Orjen and Mt. Lovcen in Montenegro) and from cultivated plants (Belgrade, Serbia) were characterized. For the latter, plants from the locus classicus, i.e., Orjenske Lokve (Mt. Orjen), were transferred to Belgrade and, after three years of cultivation, the chemical composition of their essential oils at different phenological stages was analyzed. The essential oils were obtained from the aerial parts of the plants by hydrodistillation and analyzed by GC and GC/MS. The yields and chemical compositions of the S. horvatii oils showed significant differences between the plants collected in the natural habitats and those from cultivation, as well as between the plants at different phenological stages. In the populations from the natural habitats, growing in Mediterranean conditions, the most abundant oil constituents were the phenols thymol (63.7% in the samples from Mt. Orjen) or carvacrol (68.1% in the samples from Mt. Lovcen), while the oils from the cultivated plants (Belgrade), growing in continental conditions, were dominated by linalool (up to 65.8 and 55.9% in average). The basic characteristics of the essential oil from plants at the early phenological stage (before flowering) were high percentages of linalool (37.4%), thymol (27.3%), and carvacrol (12.2%). At the stage of flowering, the percentage of linalool (56.6-57.5%) increased, while those of thymol (15.5-15.8%) and carvacrol (1.4-1.5%) significantly decreased. The essential oil of plants in the full stage of fruiting was characterized by the domination of linalool (58.4 and 65.8%) and lower percentages of thymol (7.6 and 1.3%) and carvacrol (0.7 and 0.1%). In conclusion, the oil composition of S. horvatii was found to depend on the pedoclimatic conditions of the habitat and the phenological stage of the plants.

Comparative analysis of essential oils of six Anthemis taxa from Serbia and Montenegro.

The essential-oil composition of six Anthemis taxa from several populations in Serbia and Montenegro, Anthemis triumfetti (L.) DC., A. tinctoria L., A. austriaca Jacq., A. ruthenica Bieb., A. cotula L., and A. cretica ssp. carpatica (Willd.) Grierson were analyzed by GC and GC/MS. The main constituents have been identified as follows: camphor (13.8-15.4%), alpha-pinene (5.2-9.3%), beta-pinene (4.9-7.8%), and (E)-caryophyllene (7.3-9.8%) in three populations, and cis-chrysanthenol (27.0%) and 1,8-cineole (8.4%) in one population of A. triumfetti; 1,8-cineole (9.0-25.8%) in the oils of five populations, and borneol (16.0%) and spatulenol (16.0%) in the oil of one population of A. tinctoria; cis-chrysanthenyl acetate (17.5-22.0%), beta-pinene (8.6-13.2%), and 1,8-cineole (7.2-10.4%) in the oils of A. austriaca; germacrene D (8.3-11.3%) and terpinen-4-ol (6.3-7.3%) in A. ruthenica oils; beta-cedrene (10.3-19.0%), (E)-beta-farnesene (7.8-13.5%), and germacrene D (5.2-9.1%) in the oils of A. cotula; cis-thujone (39.0%), trans-thujone (13.5%), and yomogi alcohol (7.1%) in the oil of A. cretica ssp. carpatica. The essential oil of A. austriaca was studied for the first time. A cluster analysis based on the relative percentages of all components of the essential oils was used to determine the distances between taxa and populations.

The potential of Festuca rubra and Calamagrostis epigejos for the revegetation of fly ash deposits.

Two grass species, Festuca rubra (sown) and Calamagrostis epigejos (naturally recolonised), were studied at two fly ash deposit lagoons, weathered 5 (L1) and 13 years (L2). Both species were assessed in terms of their invasive ability, photosynthetic efficiency, trace elements accumulation, and damage symptoms, while the characteristics of the habitat were assessed in terms of trace element content, and the pH and EC of the ash. In the ash weathered for 5 and 13 years, B and Mn concentrations fell within the normal range for soils, while there was an As, Mo and Cu excess, and Se and Zn deficit. There was no difference in the photosynthetic efficiency (ns) of the C. epigejos populations at the different-aged ash lagoons, but differences were found between the populations of F. rubra (P<0.001). F. rubra displayed damage symptoms, in the form of leaf tip chlorosis and necrosis, and wilting seedlings--a result of B accumulation in toxic concentrations and a Cu and Mn deficit. After 13 years the sown F. rubra remained over 9.5% of the ash lagoon's area (L2), while over the same period the naturally recolonised C. epigejos had overgrown 87.5% of the area. The greater colonisation and survival potential of C. epigejos is a result of multiple tolerance to the conditions at ash deposits and of the competitive ability of this species to dominate the colonised habitat. Therefore, characteristics of naturally colonised species can be used for modelling future actions of biological restoration of fly ash deposits aimed at binding the ash with minimal investment (the short-term aim), and for providing conditions for revegetation, and shortening the successive phases in the revegetation of ash deposits after the closure of thermal plants (the long-term aim).